Transformation of somatic embryos of soybean with chitinase and beta-1,3-glucanase genes via particle bombardment.

摘要

To transform chitinase and {dollar}beta{dollar}-1,3-glucanase genes into somatic embryos of soybean using particle bombardment, some bombardment parameters were optimized. Somatic embryos of soybean (Glycine max L.) "Jack" were bombarded with 1.0 {dollar}mu{dollar}m diameter tungsten particles coated with the plasmid pZA-300 encoding {dollar}beta{dollar}-glucuronidase (GUS) and hygromycin phosphotransferase (HPT). The plasmid was precipitated on the particles using 25 {dollar}mu{dollar}l 2.5 M CaCl{dollar}sb2,{dollar} pH 10 and 10 {dollar}mu{dollar}l 0.1 M spermidine. Other optimized parameters included the helium gas pressure, 1,100 psi; distance between rupture disk-to-target, 13 cm; distance of the rupture disk-to-macrocarrier, 8 mm and flying distance, 6 mm.; Chitinase, maize and bean chitinase and maize {dollar}beta{dollar}-1,3-glucanase gene were transformed into somatic embryos of soybean using the conditions described above. Enzyme activity assays indicated that the transformed progeny had higher activity than that of the wild type and homozygous progeny had higher activity than heterozygous progeny. Northern analysis indicated that the transgenic progeny of line 26 and 29 produced the 1.1 kb mRNA for bean chitinase. The progeny from line 29 also produced an mRNA which was larger than normal size. Pathogenic assays suggested that expression of the transgene could enhance disease resistance to brown stem rot, sudden death syndrome and brown leaf spot at the early stage of plant and disease development.